Composition including Stem Cell-Derived Exosome for Inducing Adipogenic Differentiation and Adipose Tissue Regeneration

ABSTRACT

A composition for inducing differentiation into adipocytes or regenerating adipose tissues comprises, as an ingredient, exosomes derived from stem cells differentiating into adipocytes, or exosomes derived from proliferating stem cells.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of Internationalapplication number PCT/KR2015/012013, filed Nov. 9, 2015, which claimspriority to and the benefit of Korean Patent Applications No.10-2014-0154410, 10-2015-0002660, 10-2015-0134689, and 10-2015-0137635,filed on Nov. 7, 2014, Jan. 8, 2015, Sep. 23, 2015, and September 30,the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a composition for inducing stem cellsto differentiate into adipocytes and/or regenerating adipose tissuesusing a stem cell-derived exosome containing a differentiation-inducingsubstance for the adipocytes. Further, the present invention relates toa cosmetic composition for skin whitening, wrinkle improvement or skinregeneration containing an exosome derived from stem cells.

BACKGROUND

As a therapeutic method for regenerating adipose tissues, there is amethod for using a therapeutic agent comprising stem cells, which arecultured with a matrix having an adipose tissue three-dimensionallycultured in a hydrogel. After three-dimensionally culturing the tissuein a hydrogel, a growth factor and an extracellular matrix may besecreted from the stem cells. However, to use the therapeutic agent asan injectable preparation type, it was inconvenient to remove thehydrogel in the form of a film from a culture container and to treat thetissue with a lyase. To overcome such inconvenience, a therapeuticmethod for regenerating tissues by autologous fat grafting or directlytransplanting stem cells has been developed.

In the case of autologous fat grafting, the method uses parts of thebody of a subject under operation. Therefore, the method incurs no issueof tissue or immune rejection and no observed immune response. However,the adipose tissue is highly oxygen-dependent and interacts withneighboring cells while having many blood vessels around. The graftedfat hardly exhibits a blood vessel-forming ability, and thus hasdisadvantages. For example, a cell apoptosis or cell necrosis may beinduced due to hypoxia, and the subject may need to receive severalprocedures as the rate of engraftment is not high.

Formerly, stem cells have frequently been used to restore damagedtissues that have been limited by surgery or drug therapy. For example,biopolymers such as hyaluronic acid and collagen are used for stem celltransplantation. Since stem cells can differentiate into various cellsincluding adipocytes, these stem cells have a wide range ofapplications. However, since the survival rate and the engraftment rateare low once they are placed into the body, the efficiency is reduced.Also, there is a risk that undifferentiated stem cells can form tumors.

Currently, a method for differentiating stem cells into adipocytes fortissue regeneration generally includes treating differentiation-inducingmaterials such as insulin, dexamethasone, isobutylmethylxanthine, etc.on stem cells and culturing them for a long time. However, theabove-mentioned stem cell differentiation-inducing materials areexpensive and are not effective for differentiation only by a singlecomponent. Thus, they have disadvantages. For example, they must betreated by mixing various substances, and the efficiency of celldifferentiation is low. This is problematic.

On the other hand, conventionally, attempts have been made to use aculture solution obtained by culturing stem cells as a cosmetic. Ingeneral, a culture medium containing an appropriate amount ofantibiotics and serum is used for culturing stem cells. Most of the stemcell culture solutions developed as cosmetic compositions use a normalculture medium, and a cosmetic composition including a liposome in whicha culture solution of stem cells is encapsulated in a liposome. Further,a cosmetic composition using a culture medium prepared without theingredients that are not permitted as raw materials for cosmetics and acosmetic composition containing a serum-free culture medium, and thelike have been developed.

The culture media are substances containing proteins, amino acids,hormones and growth factors for cell proliferation. The media have beenprepared in a very sophisticated manner and supplied. However, since thecell culture media, antibiotics and serum have risks that are not provenas safe, they should be used only for research purposes and their usefor the human body is prohibited. The components included in the culturemedia, such as choline chloride, hypoxanthine-sodium salt, thymidine,putrescine dihydrochloride, ferric nitrate, L-glutamine and the like,are not permitted as raw materials for cosmetics. Thus, the use of suchculture media is not suitable for a cosmetic composition. As such, theculture media contain various proteins, cytokines, growth factors andthe like secreted by stem cells. In contrast, they also containcomponents such as waste products secreted as cells grow, antibioticsadded to prevent contamination, or animal-derived serum, etc. Thus, theyare highly likely to pose various risks when used on the skin.

The components of the stem cell culture solutions to be used forcosmetics are limited. Also, during the process of encapsulating intothe liposomes, the deterioration and contamination of the components ofthe culture solutions, and an additional treatment process ofencapsulating with liposomes are required. Thus, the technique ofencapsulating the stem cell culture solutions with liposomes includinglipids to increase the skin absorption rate of the culture solutions isalso limited in the use for a cosmetic.

To complement the disadvantages of these stem cell culture solutions,techniques for using stem cell-derived exosomes have been developed.Stem cells are usually cultured in a medium containing antibiotics andserum. Bio-nanoparticles secreted from various cells present inmulticellular organisms including humans can be classified into exosomesand micro-vesicles depending on their size and difference in secretionmechanism. It is known that exosomes, which are vesicles of membranestructures secreted from various types of cells, play a variety ofroles. For example the roles include transferring membrane components,proteins, RNA, etc. by binding to other cells and tissues. Most ofsecretomes including the exosomes are obtained from a cell culturesupernatant. Thus, under a stem cell-derived exosome isolation methodcurrently used, it is difficult to completely purify the exosomes due tointerference by proteins in the medium or serum in the step of isolatingthe secretomes including the exosomes.

Accordingly, the present inventors have isolated exosomes from stemcells and discovered that the stem cell-derived exosomes have theeffects of stem cell differentiation, adipose tissue regeneration,whitening, wrinkle improvement and skin regeneration.

SUMMARY

One embodiment of the present invention provides a composition forinducing differentiation into adipocytes or regenerating adiposetissues. The composition may include, as an active ingredient, an exosome derived from proliferating stem cells, or an exosome derived fromstem cells differentiating into adipocytes.

As used herein, the “stem cells differentiating into adipocytes” referto stem cells which are differentiating into adipocytes, for example,from adipose tissue-derived stem cells (ASCs). An example of adiposetissue-derived stem cells is shown in FIG. 1. From this, the exosomescontaining genetic information, proteins and growth factors may beisolated.

Specifically, when stem cells differentiate into adipocytes, theirshapes are clearly changed, and the exosomes are isolated at this time.Therefore, it is different from isolating exosomes from undifferentiatedstem cells.

Exosomes refer to cell-derived messenger vesicles containingcell-specific components that play a role in cell-to-cell communicationby merging with a recipient cell. In an embodiment, exosomes are ofendocytic origin and are vesicles, involved in cell communication,secreted from a cell that contain cell-specific components, such aslipids, genetic material, and proteins. For example, exosomes refer tovesicles, typically 40-100 nm in size, secreted by various types ofcells and are known to carry out various roles such as transferringmembrane components, proteins, and RNA by binding to other cells andtissues. Exosomal markers include tetraspanins (CD9, CD63 and CD81) andmultivesicular body (MVB) synthesis proteins (Alix and TS G101).

The exosome may be prepared by an exosome isolation method known in theart or by the following steps, for example, 1) culturing stem cells in aculture medium, and then sub-culturing in a serum-free andnon-antibiotic medium; 2) recovering the cell culture supernatant; 3)centrifuging the recovered cell culture supernatant; and 4) separatingand purifying the exosomes, but is not limited thereto.

The stem cells differentiating into adipocytes may be bone marrow stemcells, cord blood stem cells or adipose-derived stem cells, and may behuman-, animal- or plant-derived stem cells, but are not limitedthereto.

The exosome may be used on stem cells at a concentration of 1 to 150 μgper 1 mL of the composition for inducing differentiation into adipocytesor regenerating adipose tissues, specifically at a concentration of 5 to150 μg, more specifically at a concentration of 10 to 150 μg, even morespecifically at a concentration of 20 to 130 μg, and further morespecifically at a concentration of 20 to 100 μg, but is not limitedthereto.

As used herein, the term “inducing differentiation into adipocytes”refers to the induction of stem cells to differentiate into adipocytes.

The composition may include, as an active ingredient, an exosome derivedfrom stem cells differentiating into adipocytes according to oneembodiment of the present invention. The composition may differentiatestem cells into adipocytes. Therefore, the composition may be used as acomposition for inducing differentiation into adipocytes.

As used herein, the term “regenerating adipose tissues” refers to theregeneration of adipose tissues by recovering damaged adipose tissues orinducing the production of deficient adipose tissues.

Further, the composition may regenerate adipose tissues. Therefore, thecomposition may be used as a composition for regenerating adiposetissues.

The composition for inducing differentiation into adipocytes orregenerating adipose tissues according to another embodiment of thepresent invention may be used as a pharmaceutical composition.Specifically, the pharmaceutical composition may be contained in anamount of 0.001 to 10 parts by weight based on 100 parts by weight ofthe total composition.

The pharmaceutical composition according to the above embodiment may bevarious oral or parenteral formulations. The formulations may beprepared using a diluting agent or an excipient, such as commonly-usedfillers, weighting agents, bonding agents, wetting agents,disintegrating agents, surfactants and the like. The solid formulationsfor oral administration include tablets, pills, powders, granules,capsules and the like. Such solid formulations may be prepared by mixingat least one compound with at least one excipient, for example, starch,calcium carbonate, sucrose or lactose, gelatin and the like. In additionto simple excipients, lubricants, such as magnesium stearate, talc andthe like may also be used. Liquid formulations for oral administrationinclude suspension, liquid for internal use, emulsion, syrup and thelike. In addition to commonly used simple diluents such as water andliquid paraffin, the liquid formulations may also include variousexcipients, for example, wetting agents, sweetening agents, flavoringagents, preservatives and the like. The formulations for parenteraladministration include sterile aqueous solutions, non-aqueous solvents,suspensions, emulsions, lyophilization formulations and suppositories.

As for the pharmaceutical composition according to the above embodiment,vegetable oil, such as propylene glycol, polyethylene glycol, and oliveoil, and an injectable ester, such as ethyl oleate and the like may beused as the non-aqueous solvents and suspending agents. Witepsol,macrogol, tween 61, cacao butter, laurinum, glycerol gelatin and thelike may be used as a suppository base.

The dosage forms of the pharmaceutical composition according to theabove embodiment may be in the form pharmaceutically acceptable thereof,or it may be used alone or in suitable combination with otherpharmaceutically active compounds. The salt of the exosome compound isnot particularly limited as long as it is pharmaceutically acceptable.The salt includes, for example, the salts of hydrochloric acid, sulfuricacid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid,formic acid, acetic acid, tartaric acid, lactic acid, citric acid,fumaric acid, malic acid, succinic acid, methanesulfonic acid,benzenesulfonic acid, toluenesulfonic acid, naphthalene sulfonic acidand the like.

The pharmaceutical composition according to the above embodiment may beparenterally or orally administered depending on the purpose, and may beadministered once or multiple times daily as needed such that the amountadministered is 0.1 to 500 mg, 1 to 100 mg per kg. The effective dosagefor a specific patient varies depending on the patient's body weight,age, gender, health conditions, diet, the period of administration, themode of administration, excretion rate, the severity of the disease andthe like.

According to a conventional method, the pharmaceutical compositionsaccording to the above embodiments may be used by formulating into anyform suitable for a pharmaceutical formulation including oralcompositions such as powders, granules, tablets, capsules, suspensions,emulsions, syrups, aerosols and the like, external preparations such asointments, creams and the like, suppositories and sterilized injectablesolutions.

The pharmaceutical compositions according to the above embodiments maybe administered to mammals such as rats, mice, livestock, humans and thelike using various routes such as parenteral, oral and the like, andalthough all routes of administration can be expected, it may preferablybe administered via oral, rectal or intravenous, intramuscular,subcutaneous, intrauterine, or intracerebroventricular injection.

The pharmaceutical composition for inducing differentiation intoadipocytes or regenerating adipose tissues may further includedifferentiation-inducing materials such as insulin, dexamethasone,dehydroepiandrosterone (DHEA), histamine and isobutylmethylxanthine,etc. in order to differentiate stem cells into adipocytes, but is notlimited thereto.

Another embodiment of the present invention provides a cosmeticcomposition for inducing differentiation into adipocytes or regeneratingadipose tissues and may include, as an active ingredient, an exosomederived from stem cells differentiating into adipocytes. The cosmeticcomposition may promote the regeneration of adipose tissues by inducingdifferentiation into adipocytes.

The exosome may be contained in the cosmetic composition at aconcentration of 1 to 150 μg per 1 mL of the cosmetic composition,specifically at a concentration of 5 to 150 μg, more specifically at aconcentration of 10 to 150 μg, even more specifically at a concentrationof 20 to 130 μg, and further more specifically at a concentration of 20to 100 μg, but is not limited thereto.

The cosmetic composition according to the above embodiment may containadjuvants commonly used in cosmetic or dermatological science such asfatty substances, organic solvents, solubilizing agents, thickeningagents, gelling agents, softening agents, antioxidants, suspendingagents, stabilizing agents, foaming agents, flavoring agents,surfactants, water, ionic or non-ionic emulsifiers, fillers,sequestering agents, chelating agents, preservatives, vitamins,blockers, wetting agents, essential oils, dyes, pigments, hydrophilic orlipophilic active agents, lipid vesicles or any other ingredientcommonly used in cosmetics. Such adjuvants are introduced in the amountscommonly used in the cosmetic or dermatological fields.

The external form of the cosmetic composition according to the aboveembodiment contains a cosmetically or dermatologically acceptable mediumor base. The cosmetic composition may be in any form suitable fortopical application. For example, the cosmetic composition may beprovided in the form of solutions, gels, solids, a paste, anhydrousproducts, emulsions obtained by dispersing oil phase in aqueous phase,suspensions, microemulsions, microcapsules, or ionic (liposomes) andnon-ionic vesicle dispersants, and these compositions may be preparedaccording to a conventional method in the art.

The cosmetic composition according to the above embodiment is preferablyapplied in the form of being absorbed into the skin using a microneedle,etc., but is not limited thereto.

The cosmetic composition for inducing differentiation into adipocytes orregenerating adipose tissues may include, as an active ingredient, anexosome derived from stem cells differentiating into adipocytes. Thecosmetic composition may further include differentiation-inducingmaterials such as insulin, dexamethasone, dehydroepiandrosterone (DHEA),histamine and isobutylmethylxanthine, etc. in order to differentiatestem cells into adipocytes, but is not limited thereto.

Still another embodiment of the present invention provides a mediumcomposition for stem cell differentiation which contains an exosomederived from stem cells differentiating into adipocytes and induces thestem cells to differentiate into adipocytes.

The exosome may be contained in the medium composition for stem celldifferentiation at a concentration of 1 to 150 μg per 1 mL of the mediumcomposition for stem cell differentiation, specifically at aconcentration of 5 to 150 μg, more specifically at a concentration of 10to 150 μg, even more specifically at a concentration of 20 to 130 μg,and further more specifically at a centration of 20 to 100 μg, but isnot limited thereto.

The medium composition for stem cell differentiation may further includea stem cell culture medium, but is not limited thereto.

The medium composition for stem cell differentiation may further includedifferentiation-inducing materials such as insulin, dexamethasone,dehydroepiandrosterone (DHEA), histamine and isobutylmethylxanthine,etc. to differentiate stem cells into adipocytes, but is not limitedthereto.

Further another embodiment of the present invention provides aninjectable preparation comprising a composition for inducingdifferentiation into adipocytes or regenerating adipose tissues. Theinjectable preparation may include, as an active ingredient, an exosomederived from stem cells differentiating into adipocytes; and a hydrogel.

The exosome may be contained in the injectable preparation at aconcentration of 1 to 150 μg per 1 mL, specifically at a concentrationof 5 to 150 μg, more specifically at a concentration of 10 to 150 μg,even more specifically at a concentration of 20 to 130 μg, and furthermore specifically at a concentration of 20 to 100 μg, but is not limitedthereto.

The hydrogel may be at least one hydrogel such as gelatin, alginate,chitosan, fibrin, elastin, hyaluronic acid, collagen, methyl cellulose,or collagen and methylcellulose hydrogel, but is not limited thereto.

The injectable preparation may be an injectable preparation for inducingdifferentiation into adipocytes or regenerating adipose tissues, but isnot limited thereto. That is, when the injectable preparation of thepresent invention is administered to an animal via an injection, theeffects of inducing differentiation into adipocytes or regeneratingadipose tissues may be exhibited.

In one embodiment of the present invention, the hydrogel was prepared byadding methylcellulose powder to a collagen solution. Specifically, themethylcellulose powder was added to a collagen solution dissolved in0.02 N acetic acid at a concentration of 3 mg/mL such that the finalconcentration of methylcellulose was 6% by weight. Then, the mixture wasstirred at 4° C. for 1 hour to prepare collagen and methyl cellulosehydrogel.

In one embodiment of the present invention, the injectable preparationwas prepared by carrying the exosomes, which are derived from stem cellsdifferentiating into adipocytes, in the collagen and methylcellulosehydrogel. Specifically, the exosomes derived from stem cellsdifferentiating into adipocytes were carried in the hydrogel to a finalconcentration of 50 μg/mL, and then dispersed in the hydrogel bypipetting.

The injectable preparation according to the above embodiment may beadministered to mammals such as rats, mice, livestock, humans and thelike via oral, rectal or intravenous, intramuscular, subcutaneous,intrauterine or intracerebroventricular injection.

In one embodiment of the present invention, the exosomes derived fromthe stem cells differentiating into adipocytes according to the presentinvention, as compared with the exosomes derived from proliferating stemcells, show an excellent expression rate of bioactive factors affectingthe differentiation into adipocytes (FIGS. 4 and 5).

In another embodiment of the present invention, the exosome derived fromthe stem cells differentiating into adipocytes according to the presentinvention, and the exosome derived from the proliferating stem cells, asa control group, were added in an experiment for differentiating stemcells into adipocytes. In these instances, it was confirmed that, whenthe exosomes according to the present invention were used, theadipocytes differentiated at a level similar to that of the stem cellscultured in the differentiation medium on day 7, and accordingly, oilwas produced. However, in the case of the stem cells treated with theexosomes derived from the proliferating stem cells, it was confirmedthat these stem cells only proliferated without being differentiatedinto adipocytes (FIGS. 6 and 7).

In still another embodiment of the present invention, the injectablepreparation in which the exosomes derived from the stem cellsdifferentiating into adipocytes according to the present invention werecarried in the collagen/methylcellulose hydrogel showed an excellenteffect on the regeneration of adipose tissues compared to the injectablepreparation carried with the exosomes derived from the proliferatingstem cells (FIGS. 9 and 10).

The composition for stem cell differentiation and adipose tissueregeneration according to the above embodiment includes the exosomescontaining genetic information, proteins and growth factors ofadipocytes related to the differentiation into adipocytes. Thus, thecomposition can be effectively used for the differentiation of stemcells as it is not necessary to add complex and various growth factorsfor differentiation. The stem cells differentiate into adipocytes by theexosomes derived from the stem cells differentiating into adipocytes ofthe present invention, thereby exerting an advantageous effect on theregeneration of adipose tissues when applied in vivo. The exosomederived from the stem cells differentiating into adipocytes of thepresent invention is a cell-derived material and thus is biocompatible,minimizing the side effects of the existing cell therapy agents.Further, the exosome itself can act as a carrier, which enables easyapplication of the components carried therein to the human body. Thus,the exosome can be applied as a stem cell differentiation inducer, aninjectable preparation for tissue regeneration, a filler for cosmeticpurposes, a formulation for tissue engineering and the like.

Another embodiment of the present invention provides a cosmeticcomposition containing, as an active ingredient, an exosome derived fromstem cells, and more specifically, a cosmetic composition for skinwhitening, wrinkle improvement or skin regeneration containing, anactive ingredient, an exosome derived from stem cells.

The exosome derived from the stem cell in a serum-free, andnon-antibiotic medium contains extracellular matrix derivatives as wellas collagen and growth factors effective for skin regeneration and thuscan be effectively applied for skin improvement.

In the above embodiment, the term “stem cells” refers to stem cells thatproliferate. From this, it is possible to isolate exosomes containingthe genetic information of the stem cells, proteins and growth factors.

The stem cells may be bone marrow stem cells, cord blood stem cells oradipose-derived stem cells, and may be human-, animal- or plant-derivedstem cells, but are not limited thereto.

As used herein, the term “human adipose-derived stem cells” refer tostems cells derived from human adipocytes. From this, it is possible toisolate exosomes containing the genetic information, proteins and growthfactors of the stem cells.

The method of isolating exosomes may be carried out by a method known inthe art, but is not limited thereto. In one embodiment of the presentinvention, the exosomes were isolated during the process of subculturinghuman adipose-derived stem cells. Specifically, the human adiposetissue-derived stem cells (passages 3 to 7) were cultured in a normalculture medium (Dulbecco Modified Eagle Medium, DMEM containing 10%fetal bovine serum, 1% penicillin/streptomycin). Then, at 24 hoursbefore isolating the exosomes, the cell culture media were replaced withDMEM medium, which is a serum-free and non-antibiotic medium withoutphenol red, and then maintained for 24 hours. After 24 hours, the cellculture supernatant was recovered. The recovered cell culturesupernatant was centrifuged at 300×g for 10 minutes to remove the cells,and then centrifuged at 2,000×g for 30 minutes to remove the cellsecretion. Thereafter, the cells were concentrated by centrifugation at5,000×g for 60 minutes using a centrifuge tube equipped with a filterhaving a molecular weight of 3,000. The supernatant obtained after theconcentration was mixed with an exosome isolation reagent at a ratio of1:0.5 and stored at 4° C. for one day. An exosome precipitate wasobtained by centrifugation at 10,000×g for 60 minutes, then filteredthrough a 0.22 μm filter and washed with phosphate-buffered saline(PBS). The washed exosome precipitate was centrifuged at 10,000×g for 60minutes and resuspended in PBS (FIG. 1). After recovering thesupernatant, the normal culture medium was added to the stem cells andcultured. This procedure was repeated up to passages 7 of the stemcells. The cosmetic composition was prepared using the exosomes isolatedduring the process of proliferating the stem cells up to passages 7.

The exosome may be contained at a concentration of 1 to 150 μg per 1 mLof the cosmetic composition in the cosmetic composition for skinwhitening, wrinkle improvement or regeneration, specifically at aconcentration of 5 to 150 μg, more specifically at a concentration of 10to 150 μg, even more specifically at a concentration of 20 to 130 μg,and further more specifically at a centration of 20 to 100 μg, but isnot limited thereto.

In one embodiment of the present invention, the exosomes derived fromhuman adipose-derived stem cells were used at a concentration of 10, 30or 50 μg/mL. Further, an excellent wound healing of human foreskinfibroblasts (FIG. 18), an excellent collagen synthesis rate (FIG. 19)and a decrease in melanin synthesis (FIG. 20) were confirmed.

The exosome may be contained in the cosmetic composition in the form ofa liposome encapsulating the exosome by encapsulating the exosome intothe liposome, but is not limited thereto. In some embodiments, theexosome may be in any form as long as it is suitable for use as acosmetic composition. It is also possible to use the exosome itselfwithout being encapsulated into a liposome.

When the exosome is used in the form of liposome encapsulation, theexosome may be contained in an amount of 0.1 to 10.0% by weight, morespecifically in an amount of 0.1 to 1.0% by weight based on the totalweight of the liposome, but is not limited thereto.

The liposome encapsulating the exosome may be contained in an amount of0.001 to 10.0% by weight, specifically in an amount of 0.001 to 1.0% byweight, more specifically in an amount of 0.01 to 1.0% by weight, andeven more specifically in an amount of 0.01 to 0.1% by weight based onthe total weight of the entire cosmetic composition, but is not limitedthereto.

In one embodiment of the present invention, 3% by weight of lecithin wasdispersed in an aqueous phase containing 0.01% by weight of the exosomesderived from the stem cells at room temperature (e.g., 15° C.), and thena reverse micelle emulsion (water/low temperature process carbondioxide) was formed using supercritical carbon dioxide. Then, thereaction was terminated, and the supercritical carbon dioxide wasvaporized under reduced pressure to remove the supercritical carbondioxide phase, thereby obtaining a low-temperature process liposomesuspension in which the exosomes are encapsulated. The cosmeticcomposition was prepared such that the thus-prepared liposomeencapsulating the exosomes was contained in an amount of 5% by weightbased on the total weight of the entire cosmetic composition.

In conventional technology, a culture solution obtained during theculturing of human adipose-derived stem cells was used. However, theaforesaid embodiment is different therefrom in that the exosome in theform of a nano-vesicle present in the culture solution is isolated andpurified to be used as a cosmetic ingredient, without using the culturesolution as it is. When the stem cell exosomes are isolated andpurified, the regeneration-related proteins, collagen derivatives andvarious growth factors contained in the exosomes can be effectively usedin such a mariner that they eliminate interference from mediumcomponents. Thus, the problems caused by the medium components includingantibiotics and serum can be solved.

The exosome according to the above embodiment contains the geneticinformation, protein and growth factors of the stem cells, and theexosome itself can serve as a carrier. The exosome composed of lipids ofabout 50 to 150 μm in size is biocompatible because it is a cell-derivedmaterial, and shows an excellent cell absorption rate. Therefore, it hasadvantages in that no additional processes of encapsulating the culturesolution into the liposome are necessary as in the conventionaltechnology, and that it can be easily applied to the skin.

Further, the cosmetic composition containing the exosomes derived fromthe stem cells according to the above embodiment may be used as aformulation for improving the appearance of scars. Since the stem cellderived-exosomes contain proteins and growth factors that induce cellproliferation and differentiation, and skin regeneration, they can beapplied to old wounds and acne scars to reduce scarring or theappearance thereof. Therefore, when used as a formulation for improvingscar tissue, it can be applied in the form of sprays, a gel-typeointments and patches containing the exosome derived from stem cells,etc.

In addition, still further another embodiment of the present inventionprovides a pharmaceutical composition containing, as an activeingredient, an exosome derived from stem cells, more specifically, apharmaceutical composition for skin regeneration containing, as anactive ingredient, an exosome derived from stem cells. Accordingly, thecosmetic composition for skin regeneration containing, as an activeingredient, an exosome derived from stem cells according to oneembodiment of the present invention may be used as a pharmaceuticalcomposition.

The stem cells may be bone marrow stem cells, cord blood stem cells oradipose-derived stem cells, and may be human-, animal- or plant-derivedstem cells. For example, these stem cells may be human adipose-derivedstem cells, but are not limited thereto.

In one embodiment of the present invention, when the size of the exosomederived from proliferating human adipose-derived stem cells (Stem-EXO)was examined, the size was about 69 nm, confirming that it is smallerthan the exosome derived from human epidermal keratinocytes (K-EXO) orthe exosome derived from human foreskin fibroblasts (F-EXO) (FIG. 13).

In another embodiment of the present invention, when the bioactivefactors involved in wrinkle improvement, whitening and skin regenerationpresent in Stem-EXO, K-EXO and F-EXO were compared and analyzed, it wasconfirmed that the monocyte chemoattractant protein-1, -3 (MCP-1, -3),chemokine ligand 5 (CCL-5) and collagenase inhibitor (the tissueinhibitor of metalloproteinase-1 (TIMP-1)) related to the mechanismsassociated with promoting collagen synthesis and inhibiting thedegradation thereof, interleukin-6, -8 (IL-6, -8) associated withwhitening, hepatocyte growth factor (HGF), plasminogen activatorinhibitor-1 (PAI-1), angiogenin and angiopoietin-1 associated with skinregeneration and angiogenesis were over-expressed in Stem-EXO comparedto K-EXO and/or F-EXO (FIGS. 15, 16 and 17).

In still another embodiment of the present invention, the wound healingeffect of the human foreskin fibroblasts in Stem-Exo was examined. As aresult, when Stem-EXO was used at a concentration of 10, 30 and 50μg/mL, it showed an excellent effect on the migration of foreskinfibroblasts compared to K-EXO or F-EXO, thereby showing an excellenteffect on would healing (FIG. 18).

In further another embodiment of the present invention, the wrinkleimprovement effect of Stem-EXO was confirmed. As a result, the collagensynthesis was increased as the concentration at which the Stem-EXO wasused increased. Specifically, Stem-EXO showed a remarkably excellentcollagen synthesis rate compared to K-EXO or F-EXO at 50 μg/ml, therebyconfirming an excellent effect on the wrinkle improvement (FIG. 19).

In still another embodiment of the present invention, the inhibitoryeffect of Stem-EXO on melanin formation was examined. As a result, whenStem-EXO was used on mouse melanoma at a concentration of 10, 30 and 50μg/mL, it was confirmed that the melanin synthesis was decreased,thereby confirming a remarkably excellent whitening effect (FIG. 20).

The exosomes according to embodiments of the present invention show anexcellent expression rate of bioactive factors influencing thedifferentiation into adipocytes and have the effect of differentiatingstem cells into adipocytes. Accordingly, the present invention can beapplied as stem cell differentiation inducing agents, injectablepreparations for tissue regeneration, fillers for cosmetic purposes,preparations for tissue engineering, etc. Further, the exosomesaccording to embodiments of the present invention are exosomes which aresecreted during the proliferation of stem cells and contain genes,proteins, growth factors and the like associated with cellproliferation, differentiation and regeneration of stem cells, and thuscan induce skin regeneration without other additives such as cellactivators or growth factors. Furthermore, the exosomes are purifiedcomponents which do not include antibiotics, serums or harmful factorsof a culture solution, and thus, the problems associated with culturecosmetics can be overcome. Further, the exosomes are cell-derived lipidcarriers, thereby showing excellent cell infiltration and being highlyeffective in delivering effective factors. Accordingly, the presentinvention can be applied to functional cosmetic compositions having skinwhitening, wrinkle improvement and skin regeneration functions, andpreparations for improving the appearance of scars for cosmeticpurposes, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the exosomes derived from stem cellsdifferentiating into adipocytes and the application thereof.

FIG. 2 is a schematic diagram of the method for isolating exosomes fromstem cells differentiating into adipocytes.

FIG. 3A to 3C shows diagrams illustrating the characteristics of theexosomes derived from stem cells differentiating into adipocytes; A:structure and shape of the exosomes (transmission electron microscope),B: size of the exosome (nanoparticle analyzer, dynamic lightscattering), C: exosome membrane surface marker (Western Blot).

FIG. 4A to 4C shows diagrams illustrating lipid-related bioactivefactors in the exosomes through a microarray; A: exosomes derived fromproliferating stem cells (hASC-EXO), B: exosomes derived from stem cellsdifferentiating into adipocytes (D-EXO), C: adipokine array map.

FIG. 5 shows a diagram illustrating the expression rate of factorsaffecting the differentiation into adipocytes; exosomes derived fromproliferating stem cells (hASC-EXO) and exosomes derived from stem cellsdifferentiating into adipocytes (D-EXO).

FIG. 6 shows the result of inducing differentiation of humanadipose-derived stem cells into adipocytes; A: human adipose-derivedstem cells (hASCs), B: positive control group (DM), exosomes derivedfrom stem cells differentiating into adipocytes (D-EXO) and exosomesderived from proliferating stem cells (hASC-EXO).

FIG. 7 shows the result of Oil red O staining of stem cells induced todifferentiate into adipocytes; A: human adipose-derived stem cells(hASCs), B: positive control group (DM), exosomes derived from stemcells differentiating into adipocytes (D-EXO) and exosomes derived fromproliferating stem cells (hASC-EXO).

FIG. 8 shows the result of inducing the formation of adipose tissues for3 weeks by carrying the exosomes in a hydrogel obtained by mixingcollagen and methylcellulose and subcutaneously injecting the exosomesinto nude mice models; A: collagen/methylcellulose hydrogel (Gel), B:hydrogel carrying the exosomes derived from proliferating stem cells(hASC-EXO), C: hydrogel carrying the exosomes derived from stem cellsdifferentiating into adipocytes (D-EXO).

FIG. 9 shows the result of hematoxylin-eosin staining of the gelssubcutaneously injected into nude mice models. One gel does not carrythe exosome (Gel), and the other gels carry the exosomes derived fromproliferating stem cells (hASC-EXO) and the exosomes derived from stemcells differentiating into adipocytes (D-EXO), respectively. A, C, E:40× magnification; B, D, F: 100× magnification.

FIG. 10 shows the result of Oil red O staining of the gelssubcutaneously injected into nude mice models. One gel does not carrythe exosome (Gel), and the other gels carry the exosomes derived fromproliferating stem cells (hASC-EXO) and the exosomes derived from stemcells differentiating into adipocytes (D-EXO), respectively.

FIG. 11 is a schematic diagram of the method of isolating exosomes fromproliferating human adipose-derived stem cells.

FIG. 12 shows images of human adipose-derived stem cells, humanepidermal keratinocytes, and human foreskin fibroblasts observed with amicroscope.

FIG. 13A to 13C shows diagrams illustrating the characteristics of theexosomes derived from human adipose-derived stem cells (Stem-Exo). Theexosomes derived from human keratinocytes (K-Exo) and from humanforeskin fibroblasts (F-Exo) were used as control groups. The structureand shape of the exosomes (transmission electron microscope), and thesize of the exosomes (nanoparticle analyzer, dynamic light scattering)were illustrated respectively; A: Stem-Exo (scale bars indicate 50 nm(black), 100 nm (white), respectively), B: K-Exo (scale bars indicate 50nm (black), 100 nm (white), respectively) and C: F-Exo (scale barsindicate 50 nm (black) and 200 nm (white), respectively).

FIG. 14A to 14D shows diagrams showing a comparison of the expressionlevels of bioactive factors contained in the exosomes derived from humanadipose-derived stem cells (Stem-EXO), the exosomes derived from humanepidermal keratinocytes (K-EXO) and the exosomes derived from humanfibroblasts (F-EXO) using a microarray; A: table of microarray, B:result of microarray, C and D: graph showing the relative expressionlevels of bioactive factors.

FIG. 15A to 15I shows graphs illustrating the expression level ofbioactive factor (A: PDGF-AA, B: PDGF-AB, C: PDGF-BB, D: FGF-6, E: MCP-F: MCP-3, G: Eotaxin, H: CCL-5, I: TIMP-1) related to wrinkleimprovement effect in the exosomes using a microarray; Stem-EXO:exosomes derived from proliferating human adipose-derived stem cells,K-EXO: exosomes derived from human epidermal keratinocytes, F-EXO:exosomes derived from human foreskin fibroblasts.

FIG. 16A to 16D shows graphs illustrating the expression level ofbioactive factor (A: TGF-beta, B: TNF-alpha, C: IL-6, D: IL-8) relatedto whitening effect in the exosomes using g a microarray; Stem-EXO:exosomes derived from proliferating human adipose-derived stem cells,K-EXO: exosomes derived from human epidermal keratinocytes, F-EXO:exosomes derived from human foreskin fibroblasts.

FIG. 17A to 17F shows graphs illustrating the expression levels ofbioactive factors (A: EGF, B: HGF, C: PAI-1, D: VEGF, E: Angiogenin, F:Angiopoietin-1) related to skin regeneration and angiogenesis in theexosomes using a microarray; Stem-EXO: exosomes derived fromproliferating human adipose-derived stem cells, K-EXO: exosomes derivedfrom human epidermal keratinocytes, F-EXO: exosomes derived from humanforeskin fibroblasts.

FIG. 18A to 18B shows diagrams illustrating the effect of humanadipose-derived stem cell exosomes (Stem-EXO) on the migration of humanfibroblasts; GM: stem cell culture medium (growth medium), SFM:serum-free medium, Stem-EXO: exosomes derived from proliferating humanadipose-derived stem cells, K-EXO: exosomes derived from human epidermalkeratinocytes, F-EXO: exosomes derived from human foreskin fibroblasts.

FIG. 19 shows a graph illustrating the effect of human adipose-derivedstem cell exosomes (Stem-EXO) on collagen synthesis of humanfibroblasts; SFM: serum-free medium, Stem-EXO: exosomes derived fromproliferating human adipose-derived stem cells, K-EXO: exosomes derivedfrom human epidermal keratinocytes, F-EXO: exosomes derived from humanforeskin fibroblasts.

FIG. 20 shows a graph illustrating the effect of human adipose-derivedstem cell exosomes (Stem-EXO) on the melanin synthesis of mousemelanocytes; GM: stem cell culture medium (growth medium), SFM:serum-free medium, Stem-EXO: exosomes derived from proliferating humanadipose-derived stem cells, K-EXO: exosomes derived from human epidermalkeratinocytes, F-EXO: exosomes derived from human foreskin fibroblasts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention is to provide a composition forinducing differentiation into adipocytes or regenerating adiposetissues. The composition may include, as an active ingredient, anexosome derived from stem cells differentiating into adipocytes, or anexosome derived from proliferating stem cells.

Another aspect of the present invention is to provide a cosmeticcomposition comprising a composition for inducing differentiation intoadipocytes or regenerating adipose tissues. The cosmetic composition mayinclude, as an active ingredient, an exosome derived from stem cellsdifferentiating into adipocytes.

Still another aspect of the present invention is to provide a mediumcomposition for inducing differentiation into adipocytes or regeneratingadipose tissues. The medium composition may include, as an activeingredient, an exosome derived from stem cells differentiating intoadipocytes.

Further another aspect of the present invention is to provide aninjectable preparation comprising a composition for inducingdifferentiation into adipocytes or regenerating adipose tissues. Theinjectable preparation may include, as an active ingredient, an exosomederived from stem cells differentiating into adipocytes, and a hydrogel.

Still further another aspect of the present invention is to provide acosmetic composition for skin whitening, wrinkle improvement or skinregeneration. The cosmetic composition may include, as an activeingredient, an exosome derived from proliferating stem cells.

Hereinafter, preferred embodiments are provided to help understanding ofthe present invention, but the embodiments are only for illustrativepurposes. Further, it would be apparent to those skilled in the art thatvarious modifications and alternative forms can be made within the scopeand technical idea of the present invention, and that such modificationsand alternative forms fall within the scope of the invention.

Some embodiments further relate to a method of inducing stem cellsdifferentiating into adipocytes. For example, the method may includepreparing exosomes derived from the stem cells differentiating intoadipocytes and treating stem cells with the exosomes for a predeterminedtime period.

Some embodiments further relate to a method of inducing adipose tissueregeneration using exosomes. For example, the method may includepreparing exosomes derived from the stem cells differentiating intoadipocytes, placing the exosomes in a supporting material, andadministering the supporting material to a subject, thereby regeneratingadipose tissues. In some embodiments, the supporting material includes ahydrogel.

Some embodiments further relate to a method of producing a compositioncontaining exosomes. For example, the method may include preparingexosomes derived from the stem cells differentiating into adipocytes andmixing the exosomes with any least one of stearic acid, cetyl alcohol,lanolin alcohol, liquid paraffin, Cyclomethicone, polyoxyethylenemonoolein acid ester hexanediol, glycerin, triethylamine, or carbomer.

Some embodiments further relate to a method of improving migration offoreskin fibroblasts. For example, the method may include preparingexosomes derived from proliferating human adipose-derived stem cells andtreating the foreskin fibroblasts with the exosomes, thereby improvingthe migration of the foreskin fibroblasts.

Some embodiments further relate to a method of improving collagensynthesis of foreskin fibroblasts. For example, the method may includepreparing exosomes derived from proliferating human adipose-derived stemcells and treating the foreskin fibroblasts with the exosomes, therebyimproving the collagen synthesis of the fore-skin fibroblasts.

EXAMPLE 1 Exosomes-Derived from Stem Cells Differentiating intoAdipocytes EXAMPLE 1-1 Isolation of Exosomes

In order to isolate the exosomes from stem cells differentiating intoadipocytes, the differentiation into adipocytes was induced by culturingthe stem cells in a differentiation medium.

The differentiation into adipocytes was confirmed as lipid droplets wereformed in the cytoplasm while the stem cells became gradually uneven.The differentiating stem cell culture media were replaced with aserum-free medium and maintained for 48 hours, and the cell culturesupernatant was recovered. The recovered cell culture supernatant wascentrifuged at 300×g for 10 minutes to remove the cells, and thencentrifuged at 2,000×g for 30 minutes to remove the cell secretion.

Thereafter, the cells were concentrated by centrifugation at 5,000×g for60 minutes using a centrifuge tube (molecular weight cut off=3,000,amicon tube) equipped with a filter having a molecular weight of 3,000.The supernatant obtained after the concentration was mixed with anexosome isolation reagent at a ratio of 1:0.5 and stored at 4° C. forone day. Subsequently, the cells were centrifuged at 10,000×g for 60minutes to obtain an exosome precipitate, then filtered through a filter(exosome spin column) having a molecular weight of 3,000, and washedwith phosphate-buffered saline (PBS). The washed exosome precipitate wascentrifuged at 10,000×g for 60 minutes and resuspended in PBS (FIG. 2).

EXAMPLE 1-2 Microscopic Analysis of Exosomes

The size and shape of the exosomes derived from Example 1-1 wereconfirmed using a transmission electron microscope and dynamic lightscattering, and the surface protein of the exosomes was confirmed usingWestern Blot which detects a specific protein to the membrane surface ofexosomes.

As a result, the exosomes isolated as shown in FIG. 3A were confirmed bya transmission electron microscope, and the size thereof was confirmedto be about 50.75 to 58.77 nm on average as shown in FIG. 3B. Inaddition, as shown in FIG. 3C, an exosome-specific marker expressed onthe surface of the exosome membrane was confirmed through an antibodyreaction.

EXAMPLE 1-3 Analysis of Proteins and Bioactive Factors Related toAdipocyte Differentiation in Exosomes

A microarray was used to analyze the lipid-related bioactive factorspresent in the exosomes derived from stem cells differentiating intoadipocytes and the exosomes derived from proliferating stem cells. Themicroarray was carried out through an antigen-antibody reaction, and thedegree of fluorescence (Streptavidin-Cy3) expression was measured usinga laser scanner (GenePix 4000B).

In addition, macrophage colony stimulating factor (MCSF), tumor necrosisfactor-α (TNF-α), leptin, insulin, angiopoietin 1 (ANGPT1), andadipocyte complement-related protein of 30 kDa (Acrp30), all of whichare bioactive factors influencing the differentiation into adipocytesamong the factors expressed in the microarray analysis, were confirmed,and in this regard, the relative expression levels thereof in theexosomes derived from the stem cells differentiating into adipocytes andfrom proliferating stem cells were compared.

As a result, as shown in FIGS. 4A to 4C and Table 1, it was confirmedthat there are different types of lipid-related bioactive factorspresent in the exosomes derived from the proliferating stem cells(hASC-EXO) and the exosomes derived from the stem cells differentiatinginto adipocytes (D-EXO), and it was also confirmed that there was asignificant difference in the expression levels of bioactive factorsaffecting differentiation into adipocytes (FIG. 5).

TABLE 1 hASC-EXO D-EXO Adipsin ACRP30* OPG CRP ANGPT1* PDGF-AB FasANGPTL4* SDF-1 IL-1 sRI IL-1R4/ST2 TECK IL-6 IL-10 TGF-β* MCP-1 Insulin*TIMP2 MCP-3 Leptin* TNF-α* PDGF-BB MCSF* XEDAR

EXAMPLE 1-4 Induction of Adipocyte Differentiation using Exosomes

In order to induce adipocyte differentiation of stem cells using theexosomes, medium compositions each containing the exosomes derived fromproliferating stem cell culture medium and the exosomes derived fromstem cells differentiating into adipocytes were used. The mediumcompositions were used by adding the exosomes to the stem cell culturemedium at a concentration of 30, 50 and 100 μg/mL. After treating eachmedium composition on the cultured human adipose-derived stem cells(hASCs), the medium compositions were replaced once in every 3 days for14 days.

The stem cells cultured in Dulbecco's Modified Eagle's Medium HighGlucose medium (DMEM) containing 5% fetal bovine serum, 1 μMdexamethasone, 1 μg/mL insulin, 100 μM indomethacin, 0.5 mM3-isobutyl-1-methylxanthine were used as a positive control group. Thestem cells treated with the exosomes derived from proliferating stemcells were used as a positive control group. Then, the cell shape andwhether the differentiation was carried out were analyzed with respectto the stem cells, in which the differentiation into adipocytes wasinduced, using a microscope and Oil-red O staining for 14 days.

As a result, when the exosomes derived from the stem cellsdifferentiating into adipocytes (D-EXO) were used, the stem cells weredifferentiated into adipocytes at a level similar to that of thepositive control (DM) on day 7 (FIG. 6), and accordingly, the productionof oil was confirmed (FIG. 7). However, in the case of the stem cellstreated with the exosomes derived from proliferating stem cells(hASC-EXO), it was confirmed that only proliferation was carried outwithout differentiation into adipocytes

EXAMPLE 1-5 Cosmetic Composition Comprising Exosomes Derived from StemCells Differentiating into Adipocytes

According to Example 1-1, a liposome encapsulating the exosomes -derivedfrom stem cells differentiating into adipocytes was prepared.Specifically, 3% by weight of lecithin was dispersed in an aqueous phasecontaining 0.01% by weight of the exosomes derived from stem cellsdifferentiating into adipocytes at room temperature (15° C.), and then areverse micelle emulsion (water/low temperature process carbon dioxide)was prepared using supercritical carbon dioxide. Subsequently, thereaction was terminated, the supercritical carbon dioxide was vaporizedunder reduced pressure to remove the supercritical carbon dioxide phase,and a low temperature process liposome suspension, in which the exosomesderived from stem cells differentiating into adipocytes areencapsulated, was obtained. Here, the temperature of the reactionprocess was 4° C. or below.

The cosmetic composition was prepared by the composition shown in Table2 below using the liposome encapsulating the exosomes.

TABLE 2 Content (% by Composition weight) Stearic acid 2 Cetyl alcohol 2Lanolin alcohol 2 Liquid paraffin 7 Cyclomethicone 5 Polyoxyethylenemonooleic acid ester 2 Hexanediol 2 Glycerin 3 Triethylamine 5 Carbomer0.2 Liposome encapsulating the exosomes according 0.01 to Example 1-1 ofthe present invention Purified water remainder

EXAMPLE 1-6 Induction of Adipose Tissue Regeneration using ExosomesDerived from Stem Cells Differentiating into Adipocytes

In order to confirm the effect on the adipose tissue regeneration whenthe exosomes derived from stem cells differentiating into adipocyteswere injected into the body, the exosomes derived from the proliferatingstem cells and the exosomes derived from stem cells differentiating intoadipocytes were independently carried in a collagen/methylcellulosehydrogel.

Specifically, the hydrogel was prepared by adding methylcellulose powderto a collagen solution to form the collagen/methylcellulose hydrogel.That is, methylcellulose powder was added to a collagen solutiondissolved in 0.02 N acetic acid at a concentration of 3 mg/mL such thatthe final concentration of methylcellulose became 6% by weight, and thenthe mixture was stirred at 4° C. for 1 hour to prepare the gel. In thethus-prepared collagen/methylcellulose hydrogel, the exosomes derivedfrom the proliferating stem cells or the exosomes derived from stemcells differentiating into adipocytes were carried. Specifically, theexosomes were carried in the collagen/methylcellulose hydrogel to afinal concentration of 50 μg/mL, and then dispersed in the hydrogel bypipetting. Further, the hydrogel containing the exosomes wassubcutaneously injected into the nude mice and observed for 3 weeks. Thehydrogel containing no exosome was used as a negative control group, andthe hydrogel containing the exosomes derived from proliferating stemcells (hASC-EXO) was used as a positive control group (FIG. 8). Threeweeks later, hematoxylin-eosin staining and oil red o staining wereperformed to confirm the regeneration of adipose tissues in thetransplanted hydrogel.

As a result, a large amount of mouse cells was introduced into the gelcontaining the exosomes derived from the stem cells differentiating intoadipocytes (D-EXO) (FIG. 9), and a large number of adipocytes in whichoil was produced were observed (FIG. 10), as compared with the negativeand positive control groups. From these results, it can be concludedthat the exosomes derived from stem cells differentiating intoadipocytes or the collagen/methylcellulose hydrogel carrying theexosomes were remarkably effective in inducing the regeneration ofadipose tissues.

EXAMPLE 2 Exosomes Derived from Proliferating Stem Cells EXAMPLE 2-1Isolation of Exosomes from Proliferating Human Adipose-Derived StemCells

The exosomes were isolated during the proliferation of humanadipose-derived stem cells up to passages 7. That is, the exosomes wereisolated from the proliferating human adipose-derived stem cells.

Specifically, the human adipose tissue-derived stem cells (passages 3 to7) were cultured in a normal culture medium (Dulbecco Modified EagleMedium (DMEM) containing 10% fetal bovine serum, 1%penicillin/streptomycin). Then, at 24 hours before isolating theexosomes, the cell culture media were replaced with DMEM medium, whichis a serum-free and non-antibiotic medium without phenol red, and thenmaintained for 24 hours. After 24 hours, the cell culture supernatantwas recovered. The recovered cell culture supernatant was centrifuged at300×g for 10 minutes to remove the cells, and then centrifuged at2,000×g for 30 minutes to remove the cell secretion. Thereafter, thecells were concentrated by centrifugation at 5,000×g for 60 minutesusing a centrifuge tube equipped with a filter having a molecular weightof 3,000 (molecular weight cut off=3,000, amicon tube). The supernatantobtained after the concentration was mixed with an exosome isolationreagent at a ratio of 1:0.5 and stored at 4° C. for one day. An exosomeprecipitate was obtained by centrifugation at 10,000×g for 60 minutes,then filtered through a 0.22 μm filter (exosome spin column) and washedwith phosphate-buffered saline (PBS). The washed exosome precipitate wascentrifuged at 10,000×g for 60 minutes and resuspended in PBS (FIG. 11).After recovering the supernatant, the normal culture medium was added tothe stem cells and cultured. This procedure was repeated up to passages7 of the stem cells. The exosomes isolated during the process ofproliferating up to passages 7 were used in the following experiments.In order to compare with the efficacy of the exosomes from the humanadipose-derived stem cells, the exosomes were isolated from humanepidermal keratinocytes and human foreskin fibroblasts in the samemanner by the above method (FIG. 12).

EXAMPLE 2-2 Microscopic Analysis of Exosomes

The sizes and shapes of the exosomes derived from human adipose-derivedstem cells (Stem-Exo), the exosomes derived from human epidermalkeratinocytes (K-Exo) and the exosomes derived from human foreskinfibroblasts (F-EXO) of Example 2-1 were confirmed by using atransmission electron microscope and dynamic light scattering.

As a result, the shape of each derived exosome was confirmed by atransmission electron microscope. In addition, the sizes of the exosomesderived from human adipose-derived stem cells, the exosomes derived fromhuman epidermal keratinocytes, and the exosomes derived from humanforeskin fibroblasts were about 69 nm, about 79.7 nm and about 94.6 nm,respectively, and the size of the exosomes (Stem-Exo) derived from thehuman adipose-derived stem cells was the smallest (FIGS. 13A to 13C).

Example 2-3 Analysis of Proteins and Bioactive Factors Associated withWrinkle Improvement, Whitening and Skin Regeneration in Exosomes

A microarray analysis was performed to compare and analyze the bioactivefactors associated with wrinkle improvement, whitening and skinregeneration present in the exosomes derived from human adipose-derivedstem cells (Stem-Exo), exosomes derived from human epidermalkeratinocytes (K-Exo) and exosomes derived from human foreskinfibroblasts (F-EXO). The microarray analysis was carried out through anantigen-antibody reaction, and the degree of fluorescence(Streptavidin-Cy3) expression was measured using a laser scanner(GenePix 4000B).

Through the microarray analysis, 9 bioactive factors influencing wrinkleimprovement (PDFG-AA, PDGG-AB, PDGF-BB, FGF-6, MCP-1, MCP-3, Eotaxin,CCL-5, TIMP-1), 4 whitening-related bioactive factors (TGF-beta,TNF-alpha, IL-6, IL-8) and 6 bioactive factors related to skinregeneration and angiogenesis (EGF, HGF, PAI-1, VEGF, Angiogenin,Angiopoietin-1) were confirmed and in this regard, the relativeexpression levels of each bioactive factor in the exosomes derived fromhuman adipose-derived stem cells and the exosomes derived from humanepidermal keratinocytes and from human foreskin fibroblasts werecompared (FIG. 14). FIGS. 14C and 14D show the relative expressionlevels of the bioactive factors, and the horizontal axis indicates theexosomes from human adipose-derived stem cells and the vertical axisindicates the exosomes from epidermal keratinocytes and fibroblastexosomes, respectively. In addition, the top line and the bottom linewith the middle line of the graph at the center show 1.5-foldincrease/decrease relative to the reference value, respectively. FIGS.15A to 15I show the bioactive factors related to wrinkle improvementeffect of the exosomes, FIGS. 16A to 16D show the bioactive factorsrelated to whitening effect of the exosomes, and FIGS. 17A to 17F showthe bioactive factors related to skin regeneration and angiogenesis ofthe exosomes.

As a result, as shown in FIGS. 15, 16 and 17, it was confirmed thatthere are different types of bioactive factors present in the exosomesderived from human adipose-derived stem cells (Stem-Exo), the exosomesderived from human epidermal keratinocytes (K-Exo) and the exosomesderived from human foreskin fibroblasts (F-EXO). Specifically, it wasconfirmed that the monocyte chemoattractant protein-1, -3 (MCP-1, -3),chemokine ligand 5 (CCL-5) and collagenase inhibitor (the tissueinhibitor of metalloproteinase-1 (TIMP-1)) related to the mechanismsassociated with promoting collagen synthesis and inhibiting thedegradation thereof, interleukin-6, -8 (IL-6, -8) associated withwhitening, hepatocyte growth factor (HGF), palsminogen activatorinhibitor-1 (PAI-1), angiogenin and angiopoietin-1 associated with skinregeneration and angiogenesis were over-expressed in the exosomesderived from human adipose-derived stem cells (Stem-EXO) compared toK-EXO and/or F-EXO (FIGS. 15, 16 and 17).

EXAMPLE 2-4 Effect on Migration Effect of Human Foreskin Fibroblastsusing Exosomes Derived from Proliferating Human Adipose-Derived StemCells

In order to examine the effect of the exosomes derived from humanadipose-derived stem cells on the migration of human foreskinfibroblasts, medium compositions each containing the exosomes derivedfrom the proliferating human adipose-derived stem cell (Stem-EXO), theexosomes derived from human epidermal keratinocytes (K-EXO) and theexosomes extracted from human foreskin fibroblasts (F-EXO) were used.Each of the medium compositions was prepared by adding Stem-EXO to aDMEM serum-free culture medium at concentrations of 10, 30 and 50 μg/mL,and adding K-EXO and F-EXO to a DMEM serum-free culture medium at aconcentration of 50 μg/mL, respectively. The DMEM medium containing 10%serum was used as a positive control group and the DMEM serum-freemedium was used as a negative control group. The human foreskinfibroblasts were labeled with green fluorescent dye, then seeded in a24-well plate at 1×10⁵ cells/well and cultured in a culture medium (DMEMcontaining 10% fetal bovine serum, 1% penicillin/streptomycin) for 72hours. After culturing, an artificially uniform interval of wounds wasprepared at the center of the bottom of the plate to which the cellswere adhered using a sterilized yellow tip, and the medium compositionseach containing the exosomes was applied to the cells.

As a result, the cells treated with the medium containing Stem-EXO at 24hours showed a higher degree of migration than the cells treated withnegative control, K-EXO and F-EXO, and such tendency was even moreprominent in the medium containing Stem-EXO at a concentration of 30 and50 μg/mL. After 48 hours, the migration rapidly took place in the mediumcontaining 10, 30 and 50 μg/mL of Stem-EXO, showing better (e.g.,faster, having less scaring, or less discoloration) wound healing effectcompared to the media containing K-EXO and F-EXO (FIGS. 18A and 18B).

Therefore, the exosomes derived from human adipose-derived stem cells(Stem-Exo) showed an excellent effect on the migration of human foreskinfibroblasts compared to K-EXO or F-EXO.

EXAMPLE 2-5 Effect of Exosomes Derived from Proliferating HumanAdipose-Derived Stem Cells on Wrinkle Improvement

In order to examine the effect of the exosomes derived from humanadipose-derived stem cells on the collagen synthesis of human foreskinfibroblasts, medium compositions each containing the exosomes derivedfrom the proliferating human adipose-derived stem (Stem-EXO), theexosomes derived from human epidermal keratinocytes (K-EXO) and theexosomes derived from human foreskin fibroblasts (F-EXO) were used. Themedium compositions each was prepared by adding Stem-EXO to a DMEMserum-free culture medium at concentrations of 10, 30 and 50 μg/mL, andadding K-EXO and F-EXO to a DMEM serum-free culture medium at aconcentration of 50 μg/mL, respectively. The DMEM serum-free medium wasused as a negative control group. The human foreskin fibroblasts wereseeded in a 48-well plate at 5×10⁴ cells/well and cultured in a culturemedium (DMEM containing 10% fetal bovine serum, 1%penicillin/streptomycin) for 72 hours and then washed with PBS, and themedium compositions each containing the exosomes was applied to thecells.

After completion of culturing, the culture solution of each well wasrecovered, centrifuged at 25° C. at 3,000 rpm for 10 minutes, and thenthe supernatant was taken and used for the extraction and quantificationof soluble collagen. Each well of the plate from which the culturesolution had been removed was washed with PBS. Then the cells wereseparated from the bottom of each well by applying trypsin(trypsin-EDTA), and the number of cells was measured.

Sircol collagen assay kit (Biocolor, UK) was used for the quantificationof soluble collagen. The thus-obtained supernatant was treated withTris-HCl (pH 7.6) buffer mixed with polyethylene glycol and maintainedat 4° C. for 12 hours or more. Thereafter, the resultant was centrifugedat 12,000 rpm for 10 minutes to concentrate collagen. After removing thesupernatant, 1 mL of the provided collagen adsorption dye (sircol dyereagent) was added to the collagen pellet and then cultured with shakingfor 30 minutes. The unadsorbed dye was removed by centrifugation at12,000 rpm for 10 minutes, and the pellet was washed with an acid saltbuffer. Then the dye adsorbed on the collagen was dissolved by applyingan alkali reagent, and the absorbance was measured at a wavelength of555 nm. The absorbance was substituted into the equation of the standardcurve to calculate the amount of soluble collagen in the wells to whichStem-EXO, K-EXO, F-EXO and the negative control substance were added,respectively. The calibrated amount of collagen was substituted into theequation to calculate the synthesis rate.

As a result, the soluble collagen synthesis rate for the group treatedwith Stem-EXO increased depending on the concentration of the exosomeused, compared with the negative control group (0.138 μg). Specifically,in the case of the group treated with Stem-EXO at 50 μg/mL, the amountof collagen synthesis was 2.59 μg, which was significantly increasedcompared to the same amount of K-EXO (1.4 μg) or F-EXO (0.8 μg).Therefore, it can be implied that the exosomes derived from humanadipose-derived stem cells has the effect of promoting collagensynthesis of human foreskin fibroblasts (FIG. 19).

EXAMPLE 2-6 Inhibitory Effect of Exosomes Derived from ProliferatingHuman Adipose-Derived Stem Cells on Melanin Production using MouseMelanoma

The whitening effect of the exosomes derived from human adipose-derivedstem cells (Stem-EXO), and the exosomes derived from human epidermalkeratinocytes (K-EXO) and the exosomes derived from human foreskinfibroblasts (F-EXO) as control groups were determined by the degree ofinhibition of melanin production in mouse melanoma. The melanoma cellsare cells that are derived from mouse melanoma and secrete a melaninpigment referred to as “melanin” The melanoma cells were seeded at adensity of 1×10⁵ cells/well in a 96-well plate to adhere the cells, andthen cultured for 3 days by replacing the culture medium with a mediumcontaining Stem-EXO, K-EXO and F-EXO, respectively. After 3 days, themedium was recovered and centrifuged at 4,500 rpm for 10 minutes, andthe absorbance was measured at 405 nm to calculate the amount of melaninreleased from the cells. The cells adhered to the plate were removed byapplying trypsin (trypsin-EDTA), and the number of cells was measured,followed by centrifugation to recover the cells. The cells were washedonce with PBS and centrifuged to obtain cell pellets. To the cellpellets, 1 ml of 1 N sodium hydroxide (NaOH) solution containing 10%dimethyl sulfoxide (DMSO) was added to dissolve the melanin at 80° C.for 2 hours, then the resultant was added to a 96-well plate, and theabsorbance was measured at 405 nm. The melanin was quantified using themeasured absorbance and normalized to the protein concentration of thesample to determine the concentration of the synthesized melanin.

The exosomes derived from human adipose-derived stem cells were used onthe melanoma cells at a concentration of 10, 30 and 50 μg/mL, and thedegree of melanin synthesis was examined. As a result, it was confirmedthat the melanin synthesis was reduced at all concentrations of theexosomes derived from stem cells (FIG. 20).

EXAMPLE 2-7 Cosmetic Compositions Containing Exosomes Derived fromProliferating Human Adipose-Derived Stem Cells

According to Example 2-1, a liposome encapsulating the exosomes andderived from the proliferating human adipose-derived stem cells wasprepared.

Specifically, 3% by weight of lecithin was dispersed in an aqueous phasecontaining 0.01% by weight of the exosomes derived from theproliferating stem cells at room temperature (15° C.), and then areverse micelle emulsion (water/low-temperature process carbon dioxide)was prepared using supercritical carbon dioxide. Subsequently, thereaction was terminated, the supercritical carbon dioxide was vaporizedunder reduced pressure to remove the supercritical carbon dioxide phase,and a low-temperature process liposome suspension, in which the exosomesderived from the proliferating stem cells encapsulated, was obtained.Here, the temperature of the reaction process was 4° C. or below.

The cosmetic composition was prepared by the composition shown in Table3 below using the liposome encapsulating the exosomes.

TABLE 3 Content (% by Composition weight) Stearic acid 2 Cetyl alcohol 2Lanolin alcohol 2 Liquid paraffin 7 Cyclomethicone 5 Polyoxyethylenemonooleic acid ester 2 Hexanediol 2 Glycerin 3 Triethylamine 5 Carbomer0.2 Liposome encapsulating the exosomes according 0.01 to Example 2-1 ofthe present invention Purified water remainder

FORMULATION EXAMPLE 1 Preparation of Skin Softening Cosmetic Water (SkinLotion)

The skin softening cosmetic water (skin lotion) was prepared by thecomposition shown in Table 4 below using the liposome encapsulating theexosomes derived from the proliferating stem cells obtained by themethod of Example 2-7.

TABLE 4 Content (% by Composition weight) Liposome encapsulating theexosomes according 0.01 to Example 2-1 of the present invention Ethanol10 Glycerin 3 Butylene glycol 3 Sodium hyaluronate 0.1 Triethanolamine0.1 Antioxidants 0.1 Preservatives, flavoring, coloring 0.1 Purifiedwater remainder

FORMULATION EXAMPLE 2 Preparation of Nutritive Cosmetic Water (MilkLotion)

The nutritive cosmetic water (milky lotion) was prepared by thecomposition shown in Table 5 below using the liposome encapsulating theexosomes derived from the proliferating stem cells obtained by themethod of Example 2-7.

TABLE 5 Composition Content (% by weight) Liposome encapsulating theexosomes according 0.01 to Example 2-1 of the present invention Glycerin5 Mineral oil 4 Beeswax 4 Polysorbate-60 1.5 Carboxyvinyl polymer 0.1Butylene glycol 3 Squalane 5 Triethanolamine 0.15 Preservatives,flavoring, coloring 0.1 Purified water remainder

What is claimed is:
 1. A composition for inducing differentiation intoadipocytes or regenerating adipose tissues comprising, as an ingredient,exosomes derived from stem cells differentiating into adipocytes.
 2. Thecomposition of claim 1, wherein the stem cells differentiating into theadipocyte comprise one or more of bone marrow stem cells, cord bloodstem cells, and adipose-derived stem cells.
 3. The composition of claim2, wherein the stem cells differentiating into the adipocyte arehuman-derived, animal-derived or plant-derived stem cells.
 4. Thecomposition of claim 1, wherein the exosomes are capable of inducingdifferentiation into adipocytes or regenerating adipose tissues fromstem cells after treatment of the stem cells using the exosomes at aconcentration of 1 to 150 μg per 1 mL of the composition.
 5. A cosmeticcomposition comprising the composition of claim
 1. 6. A mediumcomposition for inducing differentiation into adipocytes comprising thecomposition of claim
 1. 7. An injectable preparation comprising thecomposition of claim 1 and a hydrogel.
 8. A method of inducing stemcells differentiating into adipocytes, the method comprising: preparingexosomes derived from the stem cells differentiating into adipocytes;and treating stem cells with the exosomes for a time period.
 9. Themethod of claim 8, further comprising: culturing the stem cells in aserum-free medium for a time period.
 10. The method of claim 8, whereinthe stem cells differentiating into the adipocyte comprise one or moreof bone marrow stem cells, cord blood stem cells, and adipose-derivedstem cells.
 11. The method of claim 10, wherein the stem cellsdifferentiating into the adipocyte are human-derived, animal-derived orplant-derived stem cells.
 12. The method of claim 8, wherein theexosomes are capable of inducing differentiation into adipocytes fromstem cells after treatment of the stem cells using the exosomes at aconcentration of 1 to 150 μg per 1 mL of a composition comprising theexosomes.
 13. A method of inducing adipose tissue regeneration usingexosomes, the method comprising: preparing exosomes derived from stemcells differentiating into adipocytes; placing the exosomes in asupporting material; and administering the supporting material to asubject, thereby regenerating adipose tissues, the supporting materialincluding a hydrogel.
 14. The method of claim 13, further comprising:culturing the stem cells in a serum-free medium for a time period. 15.The method of claim 13, wherein the stem cells differentiating into theadipocyte comprise one or more of bone marrow stem cells, cord bloodstem cells, and adipose-derived stem cells.
 16. The method of claim 15,wherein the stem cells differentiating into the adipocyte arehuman-derived, animal-derived or plant-derived stem cells.
 17. Themethod of claim 13, wherein the exosomes are capable of inducingregenerating adipose tissues from stem cells after treatment of the stemcells using the exosomes at a concentration of 1 to 150 μg per 1 mL of acomposition comprising the exosomes.
 18. A method of producing acomposition containing exosomes, the method comprising: preparingexosomes derived from stem cells differentiating into adipocytes; andmixing the exosomes with one or more of stearic acid, cetyl alcohol,lanolin alcohol, liquid paraffin, Cyclomethicone, polyoxyethylenemonoolein acid ester hexanediol, glycerin, triethylamine, and carbomer.19. The method of claim 18, further comprising: culturing the stem cellsin a serum-free medium for a time period.
 20. The method of claim 18,wherein the stem cells differentiating into the adipocyte comprise oneor more of bone marrow stem cells, cord blood stem cells, oradipose-derived stem cells.
 21. The method of claim 20, wherein the stemcells differentiating into the adipocyte are human-derived,animal-derived or plant-derived stem cells.
 22. The method of claim 18,wherein the exosomes are capable of inducing differentiation intoadipocytes or regenerating adipose tissues from stem cells aftertreatment of the stem cells using the exosomes at a concentration of 1to 150 μg per 1 mL of a composition comprising the exosomes.